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Regulation of Listeria monocytogenes internalin-like genes by σB and PrfA as revealed by subgenomic microarray analyses

ABSTRACT: The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated proteins termed internalins, which are characterized by the presence of multiple leucine-rich repeats. Subsets of internalin genes have been reported previously as transcriptionally regulated by the stress responsive sigma factor B (inlA, inlB, inlC2 and inlD) and the pleiotropic transcriptional activator PrfA (inlA, inlB, inlC). To investigate contributions of σB and PrfA to internalin gene regulation, we designed a sub-genomic microarray containing two probes for each of the 24 internalin-like genes present in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic ΔsigB strain; (ii) 10403S and an isogenic ΔprfA strain; (iii) a 10403S derivative that expresses the constitutively active PrfA* (G155S) and the ΔprfA strain; and (iv) 10403S and an isogenic ΔsigBΔprfA strain. σB- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse transcriptase PCR. Statistical analyses of microarray data demonstrated that use of two probes on a microarray for a given target gene yielded more reliable transcriptional profiling information than use of a single probe. The suitability of the PrfA* strain for examining PrfA-dependent gene expression was established quantitatively by the observation that the plcA and prfA transcript levels generated by the PrfA* strain were similar to those obtained from intracellular L. monocytogenes and significantly higher than those in a wildtype strain grown in BHI broth. Among the 24 internalin-like genes examined, 4 and 6 were positively regulated by PrfA and σB, respectively, including inlA and inlB, which were positively regulated by both PrfA and σB. In summary, our findings clearly establish broad roles for both PrfA and σB in regulating L. monocytogenes internalin gene expression Keywords: Listeria monocytogenes, Internalins, sigB, PrfA, Microarrays

ORGANISM(S): Listeria monocytogenes Listeria monocytogenes 10403S

PROVIDER: GSE6421 | GEO | 2007/04/28

SECONDARY ACCESSION(S): PRJNA99491

REPOSITORIES: GEO

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Project description:The Listeria monocytogenes genome contains more than 20 genes that encode cell surface-associated proteins termed internalins, which are characterized by the presence of multiple leucine-rich repeats. Subsets of internalin genes have been reported previously as transcriptionally regulated by the stress responsive sigma factor B (inlA, inlB, inlC2 and inlD) and the pleiotropic transcriptional activator PrfA (inlA, inlB, inlC). To investigate contributions of Ï?B and PrfA to internalin gene regulation, we designed a sub-genomic microarray containing two probes for each of the 24 internalin-like genes present in the L. monocytogenes 10403S genome. Competitive microarray hybridization was performed on RNA extracted from (i) the 10403S parent strain and an isogenic Î?sigB strain; (ii) 10403S and an isogenic Î?prfA strain; (iii) a 10403S derivative that expresses the constitutively active PrfA* (G155S) and the Î?prfA strain; and (iv) 10403S and an isogenic Î?sigBÎ?prfA strain. Ï?B- and PrfA-dependent transcription of selected genes was further confirmed by quantitative reverse transcriptase PCR. Statistical analyses of microarray data demonstrated that use of two probes on a microarray for a given target gene yielded more reliable transcriptional profiling information than use of a single probe. The suitability of the PrfA* strain for examining PrfA-dependent gene expression was established quantitatively by the observation that the plcA and prfA transcript levels generated by the PrfA* strain were similar to those obtained from intracellular L. monocytogenes and significantly higher than those in a wildtype strain grown in BHI broth. Among the 24 internalin-like genes examined, 4 and 6 were positively regulated by PrfA and Ï?B, respectively, including inlA and inlB, which were positively regulated by both PrfA and Ï?B. In summary, our findings clearly establish broad roles for both PrfA and Ï?B in regulating L. monocytogenes internalin gene expression Keywords: Listeria monocytogenes, Internalins, sigB, PrfA, Microarrays For all experiments, each strain was grown in BHI at 37oC with shaking (200 rpm) to OD600=0.4, then diluted 1:100 into fresh BHI and grown to OD600=0.4. Cells were then exposed to conditions designed to induce PrfA or sB activity (0.2% charcoal or 0.3 M NaCl, respectively). Specifically, to collect RNA for identification of PrfA-dependent genes, 0.2% charcoal (â??BHI-charcoalâ??) was added to early-log phase (OD600=0.4) 10403S and DprfA cells, which were subsequently incubated with shaking for 120 min at 37Â°C. To collect RNA for identification of PrfA-and sB-dependent genes, early-log phase 10403S and DprfADsigB cells were incubated with shaking for 120 min at 37Â°C in BHI with 0.2% charcoal. NaCl was added (0.3 M final concentration) for the final 10 min (â??BHI-charcoal+NaClâ??). To collect RNA for identification of sB-dependent genes, early-log phase 10403S and DsigB cells were incubated for 120 min in BHI with NaCl added (0.3 M final concentration) for the final 10 min (â??BHI+NaClâ??). To collect RNA for identification of PrfA- dependent genes in the prfA* strain, early-log phase prfA* and DprfA cells were subsequently incubated for 120 min in BHI. After these exposures, RNA was stabilized by the addition of two volumes of RNAprotectâ?¢ (Qiagen, CA, USA). Bacterial cells were then harvested by centrifugation and stored at â??80oC for no longer than 24 h before RNA isolation. For each growth condition described above, RNA isolations were performed on three different days to provide independent biological replicates. Each set of RNA samples was used to perform competitive hybridization microarray experiments.

Project description:Listeria monocytogenes is well known to have the ability to survive and grow under a variety of stress conditions. The ability to survive and grow under osmotic stress conditions in particular appears to be important for both growth in certain foods and food associated environments as well as for host infection. To characterize the contributions of transcriptional regulators important for stress response and virulence (i.e., Sigma B - σB and PrfA), we initially analyzed three L. monocytogenes parent strains and isogenic mutants ( delta sigB, delta prfA, and delta sigB delta prfA), representing different serotypes and lineages, for their ability to grow in BHI with 11% NaCl (1.9M) at 25°C. No significant differences were observed in terms of growth between the parent strains and their respective mutants lacking prfA (i.e. delta prfA, and delta sigB delta prfA mutant strains). While for all strains, the delta sigB mutant showed a prolonged lag phase as compared to the parent strains, maximum growth rates were only reduced for the delta sigB mutant of lineage I and IV strains. Interestingly, for the serotype 1/2b strain, the delta sigB mutant reached a higher maximum cell density than the parent strain or the delta prfA mutant. Caco-2 intestinal epithelial cells invasion assays and hemolytic activity assays showed a significant role for σB in the former and for PrfA in the latter. To initially explore the mechanism that may contribute to the extended lag phase in the delta sigB mutant, microarray was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic delta sigB mutant in lag phase at 25°C in the presence of 11% NaCl. Microarray data showed significant lower and higher transcript levels for 135 and 173 genes, respectively, in the parent strain as compared to the delta sigB strains. Overall, 51 of the 173 σB up-regulated genes had previously been found among the 249 σB-dependent genes identified in a microarray study conducted in stationary phase cells, indicating that up to 122 genes may be transcribed in a σB-dependent manner during lag phase under salt stress. Notable genes that showed higher transcript levels in the parent strain include inlD (encoding an internalin protein that may be involved in virulence), sigH (encoding an alternative σ factor), glpK (encoding a glycerol kinase) and resD (encoding a two-component response regulator ResD); while, genes that showed lower transcript levels in the parent strain include dnaK (encoding a dihydroxyacetone kinase), groES (encoding a class I heat-shock protein GroES), grpE (encoding a co-chaperone GrpE) and fri (encoding a non-heme iron-binding ferritin). These data showed that although PrfA does not contribute to growth under osmotic stress at 25°C, σB does contribute to survival of L. monocytogenes under high salt conditions. Moreover, the σB-dependent transcriptome of L. monocytogenes lag phase cells under salt stress was characterized and includes previously identified as well as novel σB-dependent genes, including a number of stress response and virulence-associated genes.

Project description:In several gram-positive bacterial genera including Bacillus, Staphylococcus, and Listeria, sigma B (σB) has been identified as a stress-responsive alternative sigma factor responsible for initiating transcription of genes (the σB regulon) involved in response to stress-inducing environmental conditions. In L. monocytogenes, a foodborne pathogen of considerable threat to public health and the food industry, σB is involved in regulation of stress response and virulence gene expression. We have defined the σB regulon in L. monocytogenes during early stationary phase and under salt stress (0.3M NaCl) conditions using whole-genome microarrays, identifying 168 genes that generated ≥2.0-fold higher signals in the parental strain 10403S than in an isogenic sigB null mutant (ΔsigB), categorized into nine functional groups including stress-response genes (12), virulence genes (5), and genes related to transport (26) and metabolism (45). To gain a broader biological perspective of the σB stress response system, we applied these microarrays to Listeria innocua under the same environmental conditions. Our studies revealed 64 candidates in the L. innocua σB regulon with ≥2.0-fold higher signals in the parent than in a ΔsigB mutant; 49 of the 64 genes overlap with the L. monocytogenes σB regulon, indicating extensive overlap in σB-controlled genes between the two species. Further transcriptional analysis using TaqMan quantitative real time RT-PCR was performed for selected genes that displayed contrasting fold changes among the four microarray data sets (two stress conditions per species). We report novel members of the L. monocytogenes σB regulon, as well as the initial definition of the L. innocua σB regulon. Our comparative studies of the σB stress response systems in L. monocytogenes and L. innocua revealed features of the σB regulon that are conserved and unique to the two species. Keywords: Listeria monocytogenes, Listeria innocua, SigB regulon, salt stress, stationary phase

Project description:PrfA activity was studied in L. monocytogenes strain EGD and in an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) after growth in brain heart infusion (BHI), Luria-Bertani broth (LB) or a defined minimal medium (MM) supplemented either with one of the three PTS-carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type EGD strain in BHI and LB with either of these carbon sources, while PrfA activity was high in minimal medium in presence of glycerol but significantly reduced in presence of cellobiose. The strains expressing the prfA and prfA* gene under the prfA promoters, P1 and P2, produced equally large amounts of PrfA protein and high PrfA activity was observed in strain EGDΔprfApPrfA* under all growth conditions. In contrast, high PrfA activity in strain EGDΔprfApPrfA was only observed when this strain was cultured in BHI but not in LB or MM (in presence of either carbon source). A ptsH mutant (lacking a functional HPr) was able to grow in BHI suggesting that growth of L. monocytogenes in this culture medium is supported by carbon sources whose uptake and metabolism are independent of the PTS pathway. However, this mutant was unable to grow in LB and MM regardless which of the four carbon sources was added, suggesting that uptake of the used carbohydrates and the catabolism of glycerol depend fully on the functional common PTS pathway. Furthermore, the growth rates of L. monocytogenes are strongly reduced in presence of large amounts of PrfA protein when growing MM but less in LB and only slightly in BHI. The expression profiles of the genes encoding PTS permeases were determined in the three strains under various growth conditions. The data suggest that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases. This SuperSeries is composed of the following subset Series: GSE12143: Listeria monocytogenes EGD after growth BHI vs. LB vs. MM GSE12145: Listeria monocytogenes EGDΔprfApPrfA and EGDΔprfApPrfA* compared to the wild type strain EGD GSE12146: Listeria monocytogenes EGD and EGD-e

Project description:The foodborne pathogen Listeria monocytogenes has the ability to develop biofilm in food-processing environment, which becomes a major concern for the food safety. PrfA, a key transcriptional activator that regulates most of the known listerial virulence gene expression, has been shown to promote L. monocytogenes biofilm formation. In this study, the whole genome microarray was used to identify differentially expressed genes associated with the putative interaction between biofilm formation and PrfA in L. monocytogenes. Comparative transcriptome analyses indicated over 21.9% of the L. monocytogenes EGDe genes (627 out of 2857 predicted) were altered in their expression in biofilm cells relative to planktonic cell populations. These genes were classed into different functional categories which cover most of the biochemical functions encountered in bacterial cells, especially involved in ion transport, DNA repair, and cell wall biosynthesis based on significant enrichment of GO terms. Among them, 185 genes were identified to be associated with PrfA and biofilm formation by comparison of the whole gene expression profiles of L. monocytogenes EGDe and its M-NM-^TprfA mutant. The expression tendency of these PrfA-associated and biofilm-specific genes were mainly opposite in M-NM-^TprfA biofilm, and most of them are involved in phage-related function, membrane bioenergetics, and cell wall. Our results indicated that L. monocytogenes biofilm formation is probably controlled by the complex regulation network involved variable genes required for the different biological pathways. This regulatory network is modified in the prfA deletion mutant in order to maintain its stable biofilm lifestyle. Gene expression of planktonic cells and biofilm cells in Listeria monocytogenes EGDe and prfA isogenic deletion strain EGDeM-NM-^TprfA with cultivated in MEM and BHI for 48 hours, were mesasued using Agilent Listeria monocytogenes customized whole-genome microarray 8x15 array. Three replicates.

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Regulation of Listeria monocytogenes internalin-like genes by σB and PrfA as revealed by subgenomic microarray analyses

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